The present invention relates to methods for preventing and treating tissue injury. More specifically, the invention relates to methods for treating major organ (especially cardiac) tissue damage, particularly when associated with ischemia/reperfusion injury, sepsis, or microbial infection.
Worldwide, the World Health Organization estimates that by 2020 up to 40 percent of all deaths will be related to cardiovascular disease. In 1995, cardiovascular disease accounted for almost 15 million deaths. Since 1900, cardiovascular disease has been the number one killer in the United States in every year except 1918 (the year of the influenza pandemic), and medical costs directly related to heart disease are estimated at 30 billion dollars annually. The American Heart Association estimates that approximately 59.7 million Americans have one or more types of cardiovascular disease. Ischemic heart disease and related cardiac myopathies are the major causes of cardiac dysfunction, with ischemic heart disease causing approximately 90% of cardiac mortalities. Myocyte loss, presumably due to apoptosis, is a feature of every known type of cardiomyopathy.
Treatments for ischemic disease include aspirin, adrenoceptor blocking agents, nitrates, and angiotensin converting enzyme (ACE) inhibitors. Heparin has been administered (either intravenously or subcutaneously) in conjunction with a nonspecific fibrinolytic agent such as streptokinase, anisoylated plasminogen streptokinase activator complex (APSAC), or urokinase.
Beta-adrenergic blockers and calcium channel blockers have been shown to have some beneficial effect in the treatment of patients with chronic heart failure, which often follows ischemic cardiac tissue damage. Combinations of thrombolytic therapy and beta-adrenergic blockers, nitroglycerin, heart-rate decreasing calcium antagonists, or angiotension-converting enzyme inhibitors are currently recommended to treat patients with acute myocardial infarction. Combinations of compounds administered for the purpose of constricting arteries, increasing arterial blood pressure, and dilating veins to enhance arterial blood flow to the brain and heart, have been proposed for the treatment of patients experiencing cardiac arrest (U.S. Pat. No. 5,588,422, issued to Lurie and Gold, Dec. 31, 1996). A combination of vasopressin and an adrenergic agent, for administration to a patient suffering from cardiac arrest, has also been shown to have some benefit (U.S. Pat. No. 5,827,893, issued to Lurie and Lindner, Oct. 27, 1998).
Haikala et al. (U.S. Pat. No. 5,968,959, Oct. 19, 1999) describe use of a phospholamban inhibitor to relieve the inhibitory effect of phospholamban on cardiac sarcoplasmic reticulum Ca2+-ATPase. Araneo et al. (U.S. Pat. No. 5,977,095, Nov. 2, 1999) describe administration of a dehydroepiandrosterone (DHEA) derivative to prevent or reduce the effects of ischemia. Singh et al. (U.S. Pat. No. 5,912,019, Jun. 15, 1999) described methods of using NO donors, inhibitors of iNOS induction, and endopeptidase inhibitors to reduce ischemia/reperfusion injury. Tomaru et al. (U.S. Pat. No. 5,869,044, Feb. 9, 1999) describe the use of batroxobin (a thrombin-like enzyme derived from snake venom) to prevent or treat ischemia/reperfusion injury. Young et al. (U.S. Pat. No. 5,863,789, Jan. 26, 1999) describe the use of IL-Ira beta polypeptides and polynucleotides for treatment of cardiac ischemia. Neely (U.S. Pat. No. 6,001,842, Dec. 14, 1999) describe methods for preventing or treating ischemia/reperfusion injury in an organ by administration of a composition containing a selective A1 adenosine receptor antagonist, a P2x purinoceptor antagonist, or both.
A variety of compounds derived from red wine or grape seed have also been demonstrated to reduce ischemia/reperfusion injury (Sato, M. et al. J. Mol. Cell. Cardiol. (1999) 31(6): 1289-1297).
Despite these many and varied methods of treatment, however, significant cardiac tissue damage occurs as a result of ischemia followed by reperfusion, placing the health of thousands of individuals at risk each year. Thus, there is clearly a need for more effective agents to prevent and treat cardiac tissue injury, especially cardiac tissue injury resulting from ischemia/reperfusion.
The present invention provides a method for reducing cardiac tissue damage in a mammalian subject, particularly a human subject, comprising administering to the subject a therapeutic dosage of a (1-3)-xcex2-D-glucan. In the method of the present invention, the therapeutic dosage is administered prior to onset of ischemia, prior to onset of symptoms of viral or bacterial infection, subsequent to onset of ischemia, or subsequent to onset of symptoms of viral or bacterial infection, particularly septicemia. The method is particularly useful for bacterial infection associated with septic sequelae. The method of the present invention is also useful for preventing or treating cardiac tissue damage resulting from infection with a virus, such as human immunodeficiency virus (HIV) or human adenovirus (Ad).
In the method of the present invention, the therapeutic dosage can be administered by standard means, including orally, parenterally, intraperitoneally, and intravenously.
The invention also provides a method for treating ischemia/reperfusion injury in a body organ of a mammalian subject, comprising administering to the subject a therapeutic dosage of a (1-3)-xcex2-D-glucan. The organ may be a heart, lung, liver, or other major organ. The ischemia/reperfusion injury may result from, for example, myocardial infarction, pulmonary embolism, or traumatic injury resulting in blood loss.
In one embodiment, the invention provides an emergency care or other kit for treating ischemia/reperfusion injury in a mammalian subject, comprising a therapeutic dosage of a (1-3)-xcex2-D-glucan and a pharmaceutically acceptable carrier. The therapeutic dosage can be packaged as single dosage units or bulk packaged, and the pharmaceutically acceptable carrier can be chosen from among a tablet, caplet, capsule, intravenous fluid, or other carrier.
FIG. 1 illustrates results of scanning densitometry of autoradiograms derived from gel shift assays. Tissue samples were harvested 3 hours subsequent to cecal ligation and puncture (CLP) performed in ICR/HSD (Harlan Sprague-Dawley, Indianapolis, Ind.) mice, with n=4 per group. NF-xcexaKB and NF-IL6 nuclear binding activity in liver and lung tissue were decreased in mice pretreated with glucan phosphate (50 mg/kg, injected intraperitoneally). Results are shown as normalized integrated intensity for binding activity in liver and lung tissue taken from mice subjected to CLP and to laparotomy only (LO).
FIG. 2 illustrates results of scanning densitometry of autoradiograms derived from gel shift assays. Tissue samples were harvested 3 hours subsequent to CLP. NF-xcexaB and NF-IL6 nuclear binding activity in liver and lung tissue were decreased in mice pretreated with scleroglugan (50 mg/kg, injected intraperitoneally). Results are shown as normalized integrated intensity for binding activity in liver and lung tissue taken from mice subjected to CLP and to laparotomy only (LO).
FIG. 3 illustrates survival rate, expressed as percentage surviving (Y axis) over time course (X axis), of mice subjected to CLP with or without glucan pretreatment (nxe2x89xa720). Glucans were administered intraperitoneally (50 mg/kg) 1 hour prior to CLP surgery. Results are shown for control (CLP with no glucan treatment), glucan phosphate (glucan phosphate pretreatment followed by CLP), and scleroglucan (pretreatment with scleroglucan, followed by CLP).
FIG. 4 illustrates normalized integrated intensity for mRNA levels assessed by RT-PCR and quantified by scanning densitometry. Tissue samples were harvested 3 hours after CLP or LO. TNF-xcex1 and IL-6 mRNA levels were measured in liver and lung tissue of ICR/HSD mice (n=4) pretreated with glucan phosphate (50 mg/kg, injected intraperitoneally), or saline solution (Control) before CLP or LO.
FIG. 5 is a graphic representation of scanning densitometry quantification of autoradiograms of gel shift assays for NF-xcexaB and NF-IL6 nuclear binding activity in liver and lung tissue for ICR/HSD mice subjected to either CLP or LO. Mice were treated with glucan phosphate (50 mg/kg, administered intravenously) 15 minutes after surgery, or saline solution (Control).
FIG. 6 graphs percentage survival (Y axis) over time course (X axis) for mice subjected to CLP and subsequently treated with glucan phosphate (50 mg/kg, administered intravenously 15 minutes post surgery) or saline solution (Control). ICR/HSD mice subjected to CLP all developed polymicrobial sepsis. However, as indicated, glucan phosphate treatment significantly (P less than 0.05) increased survival rate. nxe2x89xa720 per group.
FIG. 7 is a series of graphs illustrating RT-PCR data, quantified by scanning densitometry, for TNF-xcex1 and IL-6 mRNA levels in liver and lung tissue of ICR/HSD mice subjected to LO or CLP and treated with glucan phosphate (50 mg/kg, administered intravenously) or saline solution (Control) 15 minutes post surgery. As indicated, glucan phosphate treatment suppressed hepatic TNF-xcex1 mRNA levels by 31% (P less than 0.05) relative to CLP controls. Glucan phosphate treatment suppressed lung TNF-xcex1 and IL-6 mRNA levels by 28% and 30% (P less than 0.05) relative to CLP controls.
FIG. 8 is a graph of infarct size, as determined by triphenyltetrazolium chloride (TTC) staining, in rats treated with the experimental protocol, illustrating the effects of (1-3)-xcex2-D-glucan on myocardial infarction in rats. Rats were pretreated with glucan for i one hr. before the hearts were subjected to ischemia for 45 min., followed by reperfusion for 4 hr. Areas of left ventricle (LV), risk area (RA), and infarct area (IA) were scanned and analyzed by imaging as described in the Examples. Values are expressed as meansxc2x1SEM of 8-10 rats from each group (P less than 0.05).
FIG. 9a and FIG. 9b illustrate levels of cytoplasmic IxcexaBxcex1 in rat myocardium. Cytoplasmic proteins were isolated from rat hearts that had been subjected to ischemia for 10, 15, 30, and 45 min., respectively, and analyzed by Western blot (FIG. 9a). Blots were then subjected to image analysis. Results (FIG. 9b) are expressed as meansxc2x1SEM of 5 hearts sampled at each time point for normal group (N) and sham group (S). Statistical significance is P less than 0.05 compared to normal group.
FIG. 10a and FIG. 10b are graphs of ischemia-induced NF-xcexaB nuclear binding activity in in vivo rat myocardium. Nuclear proteins were isolated from rat hearts that had been subjected to ischemia for 10, 15, 30, and 45 min., respectively, then analyzed by electromobility shift assay (EMSA) (FIG. 10a). Results of image analysis (FIG. 10b) were expressed as meansxc2x1SEM of 5 hearts sampled at each time point for normal group (N) and sham group (S). Non-specific binding is designated as xe2x80x9cnsxe2x80x9d and statistical significance is P less than 0.05 compared to normal (N).
FIG. 11a and FIG. 11b are graphs of cytoplasmic IxcexaKxcex1 protein levels in ischemic and non-ischemic areas of the heart after ischemia/reperfusion in in vivo rat hearts. Cytoplasmic proteins were isolated from rat hearts subjected to ischemia for 15 min., followed by reperfusion for 15, 30, 60, and 180 min., respectively. Isolated proteins were analyzed by Western blot (FIG. 11a) and image analysis (FIG. 11b). In the autoradiogram (FIG. 11a), xe2x80x9cIxe2x80x9d indicates ischemic area and xe2x80x9cNxe2x80x9d indicates non-ischemic area. Results were expressed as meansxc2x1SEM of 5 hearts sampled at each time point for normal group (N) and sham group (S). P less than 0.05 compared to normal (N).
FIG. 12a and FIG. 12b graphically illustrate NF-xcexaB nuclear binding activity in ischemic (I) and non-ischemic (N) rat myocardium after in vivo ischemia/reperfusion. Nuclear proteins were isolated from rat hearts which had been subjected to ischemia for 15 min., followed by 15, 30, 60, and 180 min. reperfusion, respectively. Samples were analyzed by EMSA. The graph is FIG. 12b illustrates integrated intensity, expressed as meansxc2x1SEM of 5 hearts for each time point. Normal group is designated N, sham group as S, and non-specific binding is indicated as NS. P less than 0.05 as compared to normal (N).
FIG. 13a and 13b illustrate levels of phosphorylated IxcexaKxcex1 protein in cytoplasm of rat myocardium following ischemia/reperfusion. Cytoplasmic proteins were isolated from rat hearts that had been subjected to indicated periods of ischemia and reperfusion. Levels of phosphorylated IxcexaKxcex1 protein were examined by Western blot with antiphosphor-IxcexaBxcex1 (FIG. 13a). Phosphorylated IxcexaBxcex1 proteins were undetectable in the hearts of normal (N) and sham (S) groups. Results of scanning densitometry are expressed as meansxc2x1SEM of 5 hearts sampled at each time point (FIG. 13b).
FIG. 14a shows an IxcexaK kinase (IKK) assay and FIG. 14b is a graphic representation of the results of immunoprecipitation of IxcexaK kinase (IKK) in ischemic and non-ischemic areas of rat myocardium which had been subjected to ischemia/reperfusion injury. Rat hearts were subjected to 5 min. (15xe2x80x2) and 10 min. (10xe2x80x2) of ischemia, as well as to 15 min. ischemia followed by 15 min. reperfusion (I15xe2x80x2R15xe2x80x2), as indicated. In the autoradiogram (FIG. 14a), xe2x80x9cIxe2x80x9d indicates ischemic area and xe2x80x9cNxe2x80x9d indicates non-ischemic area. Cytoplasmic proteins were isolated from each tissue sample and immunoprecipitated with anti-IKK, followed by addition of GST-IxcexaBxcex1 substrate (FIG. 14a). IKK activity was undetectable in hearts of normal (N) and sham (S) groups (FIG. 14b). Results are expressed as meansxc2x1SEM for 5 hearts at each time point.
FIG. 15 is a graphic representation of effect of glucan on loss of IxcexaBxcex1 from rat myocardial cytoplasm following I/R. Rats were pretreated with glucan (40 mg/Kg) for one hr. before being subjected to 15 min. of ischemia and 30 min. of reperfusion. Cytoplasmic proteins were isolated and levels of IxcexaBxcex1 protein determined by Western blot analysis. Results were expressed as meansxc2x1SEM for 5 hearts at each time point. P less than 0.05 as compared to normal (N).
FIG. 16 graphically demonstrates the effect of glucan on nuclear NF-xcexaB binding activity in rat myocardium subsequent to I/R. (Glucan decreases NF-xcexaB binding activity in rat myocardium subsequent to I/R). Rats were pretreated with glucan (40 mg/Kg) for one hr. before being subjected to 45 min. of ischemia (I45), as well as 15 min. ischemia followed by 30 min. reperfusion (I15R30). Nuclear proteins were isolated and analyzed by EMSA. Results are expressed as meansxc2x1SEM for 5 hearts at each time point. P less than 0.05 as compared to normal (N).
FIG. 17 depicts RT-PCR analysis of total RNA obtained from rat myocardium pretreated with glucan (40 mg/Kg) for 1 hr. prior to 15 min. ischemia and 1 hr. reperfusion. TNFxcex1 and GAPDH are labeled at right to indicate the position of the corresponding RNA. M is a 100 bp DNA ladder. As indicated, pretreatment with glucan (I15xe2x80x2R1 hr+Glucan) decreases myocardial TNFxcex1 mRNA expression after I/R.
FIG. 18 is a graphic representation of results from TTC staining of post-ischemia tissue. Glucan phosphate (40 mg/kg) was administered intravenously 5 min. after onset of ischemia. Ischemia was then continued for an additional 40 min., followed by 4 hr. reperfusion. Infarct size was determined by TTC staining. Areas of left ventricle (LV), risk area (RA), and infarct area (IA) were scanned and quantified using an imaging analyzer. Values are expressed as meansxc2x1SEM for 8-10 rats/group (P less than 0.05).
The present invention provides a method of using (1xe2x86x923)-xcex2-D-glucans to significantly reduce myocardial infarct size, decrease I/R induced NFxcexaB activation and inflammatory cytokine gene expression, and prevent cellular apoptosis, especially in the myocardium. The invention has application for treating tissue damage induced by ischemia/reperfusion, sepsis, or viral infection. The invention is especially useful for treating cardiac tissue damage prior to or following onset of ischemia/reperfusion. This method has therapeutic value for prevention of cardiac muscle damage in individuals who experience angina or who have been determined to have other risk factors for cardiac damage. Such individuals include those with HIV infection, adenovirus infection, atherosclerosis, left ventricular dysfunction (LVD), and other risk factors that have been associated with ischemia or congestive heart failure. In the method of the present invention, administration of (1-3)-xcex2-D-glucan either prior to or subsequent to onset of ischemia, or subsequent to infection, reduces infarct size, nuclear NF-xcexaB binding activity, TNFxcex1 mRNA levels, and cardiac myocyte apoptosis. (1xe2x86x923)-xcex2-D-glucans significantly reduced I/R mediated myocardial injury and apoptosis, providing a novel and clinically relevant management strategy for myocardial I/R injury as well as chronic heart failure.
This method is also useful for the treatment of I/R injury in other tissues, such as those of the lung, liver and kidney. Administration of two glucan preparations, each with different primary structures, molecular weight, polymer charge, polydispersity and branching frequency, demonstrated that glucans protect myocardium from I/R injury and define mechanisms of myocardial I/R injury which are common to other organs, such as liver, kidney, brain, and lung. Glucan treatment: 1) decreased I/R-induced NF-xcexaB activation; 2) inhibited I/R stimulation of TNFxcex1 mRNA expression; 3) markedly reduced cardiac myocyte apoptosis after I/R injury; and 4) reduced myocardial ischemic infarct size.
NF-xcexaB is present in virtually every cell type, but is retained in the cytoplasm in an inactive form bound to IxcexaB. Upon activation of the IxcexaB kinase (IKK) by a stimulus such as TNF-xcex1, IL-1, LPS, etc., IxcexaKxcex1 is phosphorylated. Once phosphorylated, IxcexaBxcex1 is recognized by a specific ubiquitin-protein ligase, and targeted for polyubiquitinylation and degradation by the 26S proteasome. IxcexaBxcex1 degradation exposes the NF-xcexaB nuclear localization sequence, and NF-xcexaB is thereby translocated to the nucleus, where it acts as a transcription factor for a variety of cellular genes, especially those involved in the inflammatory response. NF-xcexaB regulates interferon gene expression during the antiviral response, mediates cell survival signals, and modulates cellular apoptosis. NF-xcexaB regulation has been associated with a variety of human disorders, including neurodegenerative disease, cancers, arthritis, asthma, and a number of other inflammatory conditions. TNF-xcex1 induces degradation of IxcexaB, thereby activating NF-xcexaB as a transcription factor and inducing transcription of TNF-xcex1.
Glucans are polymers of glucose that are derived from yeast, bacteria, fungi, and plants. Glucans having a xcex2-(1-3)-linked glucopyranose backbone have been previously shown to activate the immune system, and it had previously been thought that, since soluble glucans enhance both the specific and non-specific immune response mechanisms, most soluble glucans would actually stimulate TNFxcex1 production. (See Jamas, et al., U.S. Pat. No. 5,783,569, Jul. 21, 1998). One of the present inventors had demonstrated the usefulness of soluble phosphorylated glucans generally for stimulating macrophage cells to produce cytotoxic and cytostatic factors against cancer cells, as well as for therapeutic and prophylactic antimicrobial applications (Williams, et al., U.S. Pat. No. 4,761,402, Aug. 2, 1988).
The therapeutic effects of (1xe2x86x923)-xcex2-D-glucan have been investigated in relation to septic sequelae, but little has been known about the mechanisms by which glucans exert their puzzling effects. For example, the inventors and others have reported that various glucans will stimulate NF-xcexaB, NF-IL6, proinflammatory, and immunoregulatory cytokine production. (Battle J et al., Biochem. Biophys. Res. Commun. (1998) 249: 499-504; Adams D S, et al., J. Leukocyte Biol. (1997) 62: 865-873). In contrast, Hara et al. (Carbohyd. Res. (1982) 110: 77-87), Hoffman et al. (Immunol. Lett. (1993) 37: 19-25), Kiho et al. (Carbohyd. Res. (1988) 142: 344-351), Masihi et al. (Int. J. Immunopharmacol. (1997) 19: 463-468) and Ukai et al. (Pharmacobio Dynamics (1983) 6: 983-990) have also reported that certain glucans exert anti-inflammatory responses. Virtually nothing has been reported about the effects of glucans on myocardial I/R injury.
In the normal host, glucan binding to the (1xe2x86x923)-xcex2-D-glucan receptor stimulates a mild inflammatory and non-specific immunostimulatory event. (Pretus H A et al., Carbohyd. Res. (1991) 219: 203-13; Battle J. et al., Biochem. Biophys. Res. Commun. (1998) 249: 499-504; Adams D. S. et al., J. Leukocyte Biol. (1997) 62: 865-873). Glucans would therefore appear to be contraindicated for systemic inflammatory response syndrome or sepsis. However, the inventors observed that pre- or post-treatment of septic mice with (1xe2x86x923)-xcex2-D-glucan decreased tissue NF-xcexaB and NF-IL6 activity as well as TNF-xcex1 and IL-6 gene expression with a correlative increase in long-term survival.
Overproduction of pro-inflammatory cytokines such as TNFxcex1, which plays a role in cardiomyocyte apoptosis, has been suggested as a mechanism for myocardial ischemia/reperfusion (I/R) injury pathogenesis and heart failure (Cain, B. S., el al. Crit. Care Med. (1999) 27: 1309-18), although ischemia/reperfusion injury has also been suggested to be caused by a rise in energy metabolism or an increase in active oxygen species by rapid re-oxygenation and the production of peroxide.
Although the pathologies associated with sepsis and ischemia/reperfusion injury share similarities, it has been difficult to determine whether those similarities extend to the cellular mechanisms by which tissue injury occurs. For example, although endotoxin is involved in sepsis and is known to elicit an immune response involving the same proinflammatory cytokines that had been suggested to produce cardiac tissue damage in I/R injury, administration of a bolus of endotoxin has actually been shown to protect the heart from I/R damage. Although sepsis generally causes decreased myocardial performance and increased immune response, induction of sepsis by administration of gram negative bacteria into the mouse dorsal subcutaneous space can give protection from I/R injury one hour after onset of I/R. (McDonough, K. H. et al., Alcohol Clin. Exp. Res. (1994) 18: 1423-1429; McDonough, K. H. et al., Shock (1994) 1: 432-437.) Thus, there remained significant questions regarding the role of immunomodulation in therapy for pathologies associated with significant tissue damage, particularly cardiac tissue damage. The present invention describes an important mechanism for reducing I/R-induced damage, and demonstrates a novel immunomodulatory role for (1-3)-xcex2-D-glucans in therapy for cardiac tissue damage.
Soluble phosphorylated glucans for use in the method of the present invention, and methods for their preparation, are described in U. S. Pat. No. 4,739,046 (DiLuzio, Apr. 19, 1988), U.S. Pat. No. 4,761,402 (Williams et al., Aug. 2, 1988), U.S. Pat. No. 4,818,752 (Williams et al., Apr. 4, 1989), U.S. Pat. No. 4,833,131 (Williams et al., May 23, 1989), U.S. Pat. No. 4,900,722 (Williams et al., Feb. 13, 1990), and U. S. Pat. No. 4,975,421 (Williams, et al., Dec. 4, 1990), each of which is incorporated herein by reference.
In the method of the present invention, (1xe2x86x923)-xcex2-D-glucans are administered systemically, for example, by oral means, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an edible carrier. The glucan preparation can be composed of a single type of glucan, such as, for example, glucan phosphate, or can be composed of a mixture of two or more glucans, chosen from among the group consisting of glucan sulphate, scleroglucan, laminarin, carboxymethylated glucan, curdulan, particulate glucan, colloidal glucan, barley glucan or oat glucan. Glucans may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into a patient""s diet by mixing with food or appropriate liquid, such as milk or juice. For oral administration, therapeutic dosages of (1xe2x86x923)-xcex2-D-glucans may be combined with one or more excipients for use in the form of ingestible tablets, troches, capsules, caplets, wafers, buccal tablets, elixirs, suspensions, syrups, and the like. The percentage of the compositions and preparations may, of course, be varied, providing an amount of active compound within the therapeutically useful compositions such that an effective dosage level of about 1 to 150 mg/kg of patient body weight, and more preferably about 25 mg/kg to about 125 mg/kg, be administered systemically (by intravenous, intraperitoneal, subcutaneous, intradermal, or intramuscular means, for example) or up to about 1 g/kg of the patient""s body weight when given orally.
Pharmaceutically acceptable vehicles, such as, for example, tablets, pills, capsules, caplets, and the like may also contain, for example, binders such as gum tragacanth, corn starch, acacia, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, or alginic acid; a lubricant such as magnesium stearate, and a sweetening agent such as sucrose, fructose, lactose or aspartame. Flavoring agents, such as, for example, fruit flavoring (cherry, orange, grape and the like), peppermint, oil of wintergreen, or other flavorings known to those of skill in the art, may also be provided as a component of the pharmaceutically acceptable vehicle for administration of (1xe2x86x923)-xcex2-D-glucans in the method of the present invention. When the unit dosage form is a capsule, it may also contain a liquid carrier, such as, for example, polyethylene glycol or a vegetable oil. Solid unit dosage forms may also contain various other materials, present as coatings. For example, capsules, caplets, or tablets may be coated with sugar, gelatin, wax, or shellac. For liquid formulations, a syrup or elixir, for example, may contain one or more (1xe2x86x923)-xcex2-D-glucans in combination with a sweetening agent such as sucrose or fructose, a preservative such as propylparaben or methylparaben, and colorings or flavorings known to those of skill in the art of pharmaceutical preparation. It is understood that any material used in preparing any bulk or unit dosage form will be pharmaceutically acceptable and substantially non-toxic when administered in the amounts provided to a mammalian, particularly a human, subject. (1xe2x86x923)-xcex2-D-glucans for use in the method of the present invention may also be provided in the form of sustained-release preparations and devices, such as microcapsules. A sustained release delivery system such as, for example, that described in U.S. Pat. No. 6,007,843 (Drizen et al., Dec. 28, 1999) may be used to provide sustained release of (1xe2x86x923)-xcex2-D-glucans for use in the method of the present invention, as can the implantable controlled release device of Ashton et al. (U.S. Pat. No. 6,001,386, Dec. 14, 1999), both incorporated herein by reference.
In a preferred method of administration, (1xe2x86x923)-xcex2-D-glucans for use in the method of the present invention are administered intravenously or intraperitoneally by infusion, or injection. Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions, for example, or sterile powders comprising (1xe2x86x923)-xcex2-D-glucans adapted for preparation of sterile injectable or infusible solutions or dispersions. Solutions containing (1xe2x86x923)-xcex2-D-glucans can be prepared in water, preferably containing isotonic agents such as sugars or appropriate salts, such as sodium chloride, optionally admixed with a nontoxic surfactant. Dispersions of (1xe2x86x923)-xcex2-D-glucans for administration in the method of the present invention can also be prepared, for example, in glycerol, triacetin, liquid polyethylene glycols, oils, or mixtures thereof. Intravenous or intraperitoneal solutions or dispersions are also provided with a preservative formulated to prevent the growth of microorganisms within the intravenous or intraperitoneal solution or dispersion.
Sterile preparations may include (1xe2x86x923)-xcex2-D-glucans encapsulated within liposomes. In any dosage form chosen for administration, the final dosage form must be sterile, fluid, and stable under standard conditions of manufacture and storage. The liquid carrier or vehicle can comprise, for example, water, ethanol, a polyol (glycerol, propylene glycol, liquid polyethylene glycols, for example) vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. Preferably, such liquid carriers or vehicles will include isotonic agents, such as, for example, sugars, buffers or sodium chloride. Aluminum monostearate or gelatin, for example, may be incorporated with the liquid carrier or vehicle in order to prolong absorption of the injectable composition, particularly the (1xe2x86x923)-xcex2-D-glucans.
A preferred method for preparing sterile injectable solutions is combination of the (1xe2x86x923)-xcex2-D-glucans in the required amounts with various of the other ingredients described above, as required for the desired formulation, followed by filter sterilization. Vacuum drying or freeze drying techniques can be used to prepare sterile powders for the preparation of injectable solutions.
The method of the present invention provides a therapy for ischemia/reperfusion injury, especially in cardiac muscle tissue. Injury can be ameliorated by administering to a patient a dosage of from about 1 mg/kg body weight to about 150 mg/kg body weight of a (1-3)-xcex2-D-glucan such as those described in U.S. Pat. No. 4,739,046 (DiLuzio, Apr. 19, 1988), U.S. Pat. No. 4,761,402 (Williams et al., Aug. 2, 1988), U.S. Pat. No. 4,818,752 (Williams et al., Apr. 4, 1989), U.S. Pat. No. 4,833,131 (Williams et al., May 23, 1989), U.S. Pat. No. 4,900,722 (Williams et al., Feb. 13, 1990), and U.S. Pat. No. 4,975,421 (Williams, et al., Dec. 4, 1990). Glucans are effective for decreasing tissue injury when administered either prior to onset of ischemia or subsequent to onset of ischemia. Onset of ischemia can be determined by those of skill in the medical arts, by observation of symptoms associated with ischemia, such as chest pain, pressure-like discomfort, radiation of discomfort and an abnormal electrocardiogram. Less typical symptoms include diaphoresis, palpitations, nausea, dyspnea and syncope. Glucans can be administered using any of the methods and compositions previously described, a preferred method using a glucan preparation suitable for intravenous administration.
Since ischemia-reperfusion injury may follow trauma injury and most likely will occur following cardiac arrest, the present invention also provides bulk-packaged or individually-packaged injectable or intravenous solutions suitable for administration by emergency medical personnel at the site of trauma injury or cardiac arrest, as well as sterile powders suitable for preparing intravenous solutions.
Tumor necrosis factor (TNFxcex1) is involved in dilated cardiomyopathy, myocyte apoptosis, transmural myocarditis, and biventricular fibrosis. TNFxcex1 has also been proposed to play a role in the pathology of congestive heart failure. In patients with chronic heart failure, decreased plasma TNFxcex1 levels have been shown to improve heart function (Liu, L. el al., Int. J. Cardiol. (1999) 69(1): 77-82; Oral, H. et al., Clin. Cardiol. (1995) 18(9 Suppl. 4) IV 20-27). The inventors have demonstrated that (1xe2x86x923)-xcex2-D-glucans significantly decrease TNFxcex1 levels, providing a novel therapy for congestive heart failure and related cardiomyopathies.
In patients who experience congestive heart failure, heart function can be improved by administration of one or more (1xe2x86x923)-xcex2-D-glucan(s) in the method of the present invention, the dosage being administered intravenously, intramuscularly, intraperintoneally, subcutaneously, intradermally, or orally in an amount effective to deliver from about 1 mg/kg to about 150 mg/kg of the one or more (1xe2x86x923)-xcex2-D-glucans to the patient. Appropriate protocols for glucan administration to a patient experiencing congestive heart failure should be determined by the patient""s individual physician. Dosage calculation and timing are determined by techniques known to those of skill in the medical arts.
Viral infection has been associated with a variety of sequelae resulting in tissue damage. For example, a high rate of unexpected left ventricular (LV) dysfunction has been observed in human immunodeficiency virus (HIV)-infected patients (Herskowitz et al., Am. J. Cardiol. (1993) 71: 955-958), and myocardial involvement has been associated with early stage HIV infection (Coudray, N. et al. Eur. Heart J. (1995) 16: 61-67). Pathogenesis has also been associated with the inflammatory response to the virus. For example, NFxcexaB has been associated with virus-induced apoptosis of cells, and treatment of cells with oligonucleotide decoys that bind NFxcexaB have been shown to protect AT-3 cells from Sindbis virus-induced apoptosis (Lin, K-I et al. J. Cell Biol. (1995) 131: 1149-1161). TNFxcex1 induction associated with reovirus, murine hepatitis virus, and murine cytomegalovirus has been demonstrated to play a pathogenic role in the development of liver disease. Studies have demonstrated that the inflammatory responses can be separated from conditions of significant hepatic damage at early times during viral infections and have shown that endogenous cytokine contributes to virus-induced liver disease (Orange, J. S. et al., J. Virol. (1997) 71(12): 9248-9258). Activated NF-xcexaB has been shown to be required for reovirus-induced apoptosis (Connolly, J. et al., J. Virol (2000) 2981-2989). Other viruses also induce NF-xcexaB activation. For example, the long terminal repeat (LTR) of human immunodeficiency virus (HIV) contains xcexaB response elements, and activation of NF-xcexaB has been shown to directly stimulate viral gene expression (Chen, B. K. et al., J. Virol. (1997) 71: 5495-5504; Chene, L. et al., J. Virol. (1999) 73: 2064-2073). The human T-cell leukemia Tax protein induces NF-xcexaB activation (Beraud, C. and W. Green, J. Acquired Immunodef. Syndr. (1996) 13(Suppl. 1): S76-S84), while NF-xcexaB activation induces expression of cellular genes that promote HTLV replication (Ballard, D. W. et al., Science (1988) 241: 1652-1655).
The present method provides a therapy for decreasing virus-induced tissue damage by decreasing levels of NFxcexaB and TNFxcex1 which have been associated with tissue pathology. As TNFxcex1 is generally induced at early times post-infection, it is preferable to administer a dosage of from about 1 mg/kg body weight to about 150 mg/kg body weight to a patient at early stages of viral infection. The dosage can be administered systemically, by intravenous, oral, or parenteral means, or can be administered locally by injection at or near the site of infection.
The method of the present invention provides (1xe2x86x923)-xcex2-D-glucan administration to reduce tissue damage, mediated by pro-inflammatory cytokines such as NFxcexaB and TNFxcex1, during bacterial infection and especially during sepsis. Glucans are administered upon diagnosis of bacterial infection, particularly where the bacterial species is known to produce systemic infection or sepsis. Such bacterial species include, for example, Staphylococcus aure us, Pseudomonas aeruginosa, Escherichia coli, Bacteroides fragilis and Yersinia enterocolitica. Glucans can also be administered post-onset of sepsis to reduce septic sequelae, such as tissue necrosis. A dosage sufficient to deliver from about 1 mg/kg body weight to about 150 mg/kg body weight is administered either orally, parenterally, or intravenously to a patient with bacterial infection or sepsis.
All forms of transplantation involve some ischemic and traumatic injury to the donor tissue, which has been proposed as one of the reasons for the early timing of most rejection episodes. By decreasing levels of NFxcexaB and TNFxcex1 post-transplant, the donor tissue can be protected from much of the host immuneresponse involved in rejection. In the method of the present invention, a dosage sufficient to delivery from about 1 mg/kg body weight to about 150 mg/kg body weight is administered either orally, parenterally, or intravenously to a patient prior to or following organ transplantation. A preferred method of administration is intravenous administration.
An added benefit of the method of the present invention for potential heart transplant patients with congestive heart failure is the increased cardiac function associated with xcex2-glucan administration. By improving cardiac function pre-transplant, xcex2-glucan therapy can prolong the life of a CHF patient, while decreasing the probability of rejection once the donor heart is transplanted.
Inhibition of TNFxcex1 has been demonstrated to decrease inflammation and prolong adenovirus gene expression in lung and liver in gene therapy protocols (Zhang, H. G. et al. (1998) 9 (13): 1875-1884). Results from these studies demonstrated that TNFxcex1 is the primary factor involved in elimination of adenovirus-infected cells in liver and lung. Results also indicated that inhibition of TNFxcex1 prolonged the viral infection, allowing more time for delivery of the target gene. The method of the present invention provides a novel therapeutic method for prolonging effective gene therapy by decreasing TNFxcex1 levels using (1xe2x86x923)-xcex2-D-glucan administration.
In the method of the present invention, (1xe2x86x923)-xcex2-D-glucan administration is begun at onset of the gene therapy protocol (infection with the viral vector). A dosage of from about 1 mg/kg body weight to about 150 mg/kg body is administered either orally, parenterally, or intravenously. A preferred route of administration is intravenous administration. The (1xe2x86x923)-xcex2-D-glucan dosage can be delivered either systemically or locally, although the preferred method of administration is systemic administration.
Spinal cord ischemia with resulting paraplegia can result from transient occlusion of the thoracic aorta, and studies have shown that ischemia preconditioning can protect against paraplegia (Abraham, V. S. et al., Ann. Thorac. Sure. (2000) 69(2): 475-479.) The present invention provides a method for preventing and treating spinal cord injury resulting from ischemia. A therapeutic dosage of (1xe2x86x923)-xcex2-D-glucan, as described previously, can be delivered by means known to those of skill in the art, including intravenous or oral administration, as well as by direct injection into the injured tissue. Delivery of the appropriate therapeutic dosage can be made after onset of ischemia, or, especially in the case of surgical occlusion of the thoracic aorta, prior to onset of ischemia.
Ischemia of the tissues of an arm or leg in the human, or a fore- or hindlimb in a mammal, can also result from dissection or occlusion of an artery which supplies blood to those tissues. The present invention provides a means for decreasing ischemic injury to the limb tissue by providing a therapeutic dosage of (1xe2x86x923)-xcex2-D-glucan to the tissue either prior to onset of ischemia, particularly where it is a requirement for a surgical procedure, or after onset of ischemia, which may accompany traumatic injury. The therapeutic dosage can be delivered intravenously, orally, parenterally, or by direct injection at the site of the ischemic injury.